Gestational diabetes mellitus (GDM), defined by maternal hyperglycemia, affects 7% of pregnancies. GDM results in fetal overgrowth, or macrosomia, which adversely influences offspring health including greater risk of obesity. Currently, medical nutrition therapy is an integral part of the GDM care process which regulates macronutrient intake and weight gain to normalize maternal blood glucose. However, attenuating maternal hyperglycemia cannot sufficiently address the increased placental lipid transfer and fetal lipid accretion, which are the major causes of macrosomia. Choline is an essential micronutrient, whose requirement increases substantially during pregnancy. The PI has previously shown that prenatal choline supplementation in humans leads to lower fetal stress and better vascular development of the placenta. Our pilot data suggests that choline supplementation in pregnant mice activates the peroxisome proliferator-activated receptor alpha pathway, which may increase fatty acid breakdown. Choline supplementation also prevents fatty liver disease in humans and animals. We hypothesize that maternal choline supplementation will prevent GDM-induced macrosomia and excessive adiposity across the lifespan of the offspring. In Aim 1, we will determine the effects of maternal choline intake on fetal growth and lipid homeostasis during gestation in a mouse model of GDM. Six-week-old C57BL/6J mice (n=6/group/time point) will be fed either a high fat diet to induce GDM during pregnancy or a control diet. Three levels of choline bitartrate (0.25, 0.5 or 1 mg/kcal of diet) will be added to he diets to generate choline deficient, control or supplemented subgroups. We will assess whether maternal choline intake decreases placental and fetal weight and reduces fat accumulation at mid-gestation and 2.5 days preterm. In Aim 2, we will determine the effects of maternal choline intake on long-term body weight regulation and adiposity in offspring affected by GDM. C57BL/6J pups (n= 2 pups/ dam/ treatment) from GDM or control dams (n=6 dams) with varied choline intake during gestation (same as Aim 1) will be fed either a low fat diet or a high fat die (obesogenic) for 6 weeks after weaning, in order to assess whether prenatal choline supplementation reduces weight gain and adiposity of the offspring in a control or obesogenic postnatal environment. At the conclusion of these studies, we will have delineated the efficacy of choline in preventing GDM macrosomia and excessive adiposity of offspring. Results of the study will lay the ground work for human studies that use choline as an innocuous and cost effective intervention for GDM.